Functional Study On Drought And Salt-alkali Tolerance Of AfZFP5 Gene In Amorpha Fruticosa

Soil salinization is becoming increasingly serious,which seriously restricts the development of China’s agriculture.Amorpha fruticose is highly adaptable to adverse environments and is an important resource for tolerance.This study is based on transcriptome sequencing data from Amorpha fruticose and clones zinc finger protein gene 5（AfZFP5）which is in response to drought-treated.The AfZFP5 gene was integrated into the genome of tobacco by infection and transformation of Agrobacterium,and the tolerance function of AfZFP5 gene was analyzed by transgenic tobacco as experimental material.1.In this study,the zinc finger protein gene（registration number:MT313927）was successfully cloned from Amorpha fruticose,the gene open reading frame sequence is 705bp,encoding 234 amino acids,containing ZnF＿C2H2 domains,and the molecular weight of the presumed protein is 26.9KD,and the isoelectric point of the protein is 7.56,which is named AfZFP5.Phylogenetic tree analysis showed that AfZFP5 was most closely related to TpZFP5of Trifolium pratense and CaZFP5 of Cicer arietinum.2.After real-time quantitative fluorescence PCR analysis,it was found that AfZFP5 gene was expressed in the roots,stems,leaves,flowers and spikes of Amorpha fruticose,and the expression was highest in the roots and the lowest in the stems.ABA,PEG6000,NaCl,NaHCO₃,H₂O₂ treatment and three exogenous hormones,BR,IAA,SA can upregulate the expression level of AfZFP5 gene.3.The recombinant plasmid PBI121-AfZFP5-GFP was constructed and subcellular localization analysis was performed by tobacco injection,and the results showed that the place where AfZFP5 protein functioned was the nucleus,which was consistent with the prediction results of online software.4.The recombinant plasmid pYES2-AfZFP5 was constructed,and it was found that AfZFP5 yeast had stronger tolerance to Sorbitol,ABA,NaCl,NaHCO₃ and H₂O₂ treatment,which preliminarily indicated that AfZFP5 gene has drought resistance and salinity tolerance.5.Through Agrobacterium-mediated tobacco genetic transformation technology,the AfZFP5 tobacco that can stabilize the heredity was successfully obtained.Combined with phenotypic differences,related physiological indexes and FluorCam fluorescence imaging techniques,it was found that AfZFP5 tobacco had stronger tolerance than wild-type tobacco,and the integration of AfZFP5 gene played a role in improving drought resistance and salinity tolerance,and AfZFP5 tobacco in the trophic stage had a certain tolerance to low temperature stress.It is speculated that AfZFP5 gene may be related to enhancing the antioxidant capacity of plants and maintaining photosynthesis.This study has preliminarily analyzed the function of AfZFP5 gene,which will provide a theoretical basis for the study of the stress resistance mechanism of Amorpha fruticose.At the same time,it also provides gene donors for the cultivation of new varieties of tolerance,and proposes a new idea of changing plant tolerance by applying exogenous hormones to induce the expression level of resistance function genes.